Date of Award

2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biochemistry & Molecular Biophysics

First Advisor

Ben E. Black

Abstract

The accurate segregation of chromosomes during mitosis is essential for the survival and development of all eukaryotes, and this process requires the attachment of mitotic spindle microtubules to the kinetochore, which is assembled on the chromosomal centromere. Defections in centromere or kinetochore function can lead to the loss of genetic material during cell division, which can result in development defects or disease. Therefore, elucidating how the centromere is stably and accurately propagated across cell and organismal generations is crucial to our understanding of how genetic information is accurately inherited. Centromere location is specified epigenetically by the histone H3 variant termed centromere protein A (CENP-A). CENP-A molecules at the centromere are known to possess a remarkably stability, exhibiting almost no detectable turnover. This stability is crucial for maintenance of centromere identity, but the molecular basis for this stability is unclear. Additionally, new CENP-A molecules must be assembled onto centromeric chromatin at every cell cycle, or else this epigenetic mark will be diluted and inevitably lost over time. The process of CENP-A assembly is exquisitely regulated, but is poorly understood. In this thesis, we identified the role of an essential centromeric protein, CENP-C, in not only binding to CENP-A nucleosomes, but also reshaping and stabilizing it at centromeres. We then pinpoint the mechanism by which CENP-C stabilizes CENP-A nucleosomes through a critical arginine anchor, which drives the structural transition of the CENP-A nucleosome. We then assemble a core centromeric nucleosome complex (CCNC) containing the CENP-A nucleosome bound to the nucleosome-binding domains of both CENP-C and another centromeric protein, CENP-N, and provide the first biophysical insight into how both proteins collaborate to rigidify CENP-A nucleosomes in vitro. Additionally, using gene-editing and rapid protein degradation approaches, we demonstrate that CENP-C and CENP-N are both crucial in the maintenance of CENP-A nucleosomes in cells. And finally, we report the landscape of Cdk regulation on the CENP-A-specific chaperone, HJURP, which provide insight into the mechanism of nascent CENP-A assembly. Taken together, these findings advance our understanding in how centromere location is specified and maintained to ensure faithful inheritance of genetic information across cell divisions.

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